Cholera is an acute diarrheal disease caused by the gram-negative bacterium, Vibrio cholerae. Following ingestion of contaminated food or water, bacteria colonize the small intestine and secrete cholera toxin, which is responsible for the extensive loss of fluid and electrolytes from infected individuals. Cholera remains a worldwide problem. Although a number of live, attenuated or killed whole cell vaccine formulations have been tested, none have proven successful enough to result in their widespread use. This grant proposal focuses on new approaches to develop a cholera subunit vaccine formulation based on the current understanding of V. cholerae colonization, pathogenesis, and human immune responses to infection. The strategy will incorporate features of the highly successful Haemophilus influenzae type b and pertussis vaccines that utilize defined surface virulence determinants, colonization factors, and toxoids to achieve long-lasting protection. The present proposal will focus on formulations that include the toxin coregulated pilus (TCP) colonization factor, detoxified LPS, and the adjuvant cholerae toxin. The general goal for these studies is to define the most effective combination of TCP and LPS antigens that provide for protective humoral responses. We will characterize in detail the B cell epitopes for these antigens and determine which epitopes are minimally required for effective immunity. Studies will extend this information to human sera and demonstrate that the epitopes we have identified are operational in the field. Using a well- established mouse model, we will quickly be able to define the immunization regimen and immunogens that are likely candidates for extension into human trials. The proposed research formally brings together the expertise from two research groups at Dartmouth Medical School. Dr. William Wade is a molecular immunologist with training in structural and functional analyses of proteins as they relate to antigen presentation. His research focuses on methodologies to optimize the immunogenicity and delivery of antigens. Dr. Ronald Taylor's research focuses on the molecular basis of V. cholerae pathogenesis. He discovered TCP and his group has been instrumental in characterizing the tcpA gene and corresponding pilin protein as well as demonstrating that TCP is the major V. cholerae colonization factor and a protective antigen. Together, the expertise of these two research groups will provide a unique opportunity to develop and evaluate V: cholerae subunit vaccines using new approaches.